Photodynamic Inactivation of Conidia of the Fungi Metarhizium anisopliae and Aspergillus nidulans with Methylene Blue and Toluidine Blue

Antimicrobial photodynamic treatment (PDT) is a promising method that can be used to control localized mycoses or kill fungi in the environment. A major objective of the current study was to compare the conidial photosensitization of two fungal species (Metarhizium anisopliae and Aspergillus nidulans) with methylene blue (MB) and toluidine blue (TBO) under different incubation and light conditions. Parameters examined were media, photosensitizer (PS) concentration and light source. PDT with MB and TBO resulted in an incomplete inactivation of the conidia of both fungal species. Conidial inactivation reached up to 99.7%, but none of the treatments was sufficient to achieve a 100% fungicidal effect using either MB or TBO. PDT delayed the germination of the surviving conidia. Washing the conidia to remove unbound PS before light exposure drastically reduced the photosensitization of A. nidulans. The reduction was much smaller in M. anisopliae conidia, indicating that the conidia of the two species interact differently with MB and TBO. Conidia of green and yellow M. anisopliae mutants were less affected by PDT than mutants with white and violet conidia. In contrast to what occurred in PBS, photosensitization of M. anisopliae and A. nidulans conidia was not observed when PDT was performed in potato dextrose media.

New SMS mutation leads to a striking reduction in spermine synthase protein function and a severe form of Snyder-Robinson X-linked recessive mental retardation syndrome

We report the identification of a novel mutation at a highly conserved residue within the N-terminal region of spermine synthase (SMS) in a second family with Snyder-Robinson X-linked mental retardation syndrome ( OMIM 309583). This missense mutation, p.G56S, greatly reduces SMS activity and leads to severe epilepsy and cognitive impairment. Our findings contribute to a better delineation and expansion of the clinical spectrum of Snyder-Robinson syndrome, support the important role of the N-terminus in the function of the SMS protein, and provide further evidence for the importance of SMS activity in the development of intellectual processing and other aspects of human development.

Evaluation of Trypsin Treatment on the Inactivation of Bovine Herpesvirus Type 1 on In Vitro Produced Pre-implantation Embryos

The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 mu l of BoHV-1, Los Angeles strain 10(7.5) TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.

Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-d pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

Cre recombinase-mediated inactivation of H-2Dd transgene expression: evidence for partial missing self-recognition by Ly49A NK cells.

We have established H-2D(d)-transgenic (Tg) mice, in which H-2D(d) expression can be extinguished by Cre recombinase-mediated deletion of an essential portion of the transgene (Tg). NK cells adapted to the expression of the H-2D(d) Tg in H-2(b) mice and acquired reactivity to cells lacking H-2D(d), both in vivo and in vitro. H-2D(d)-Tg mice crossed to mice harboring an Mx-Cre Tg resulted in mosaic H-2D(d) expression. That abrogated NK cell reactivity to cells lacking D(d). In D(d) single Tg mice it is the Ly49A+ NK cell subset that reacts to cells lacking D(d), because the inhibitory Ly49A receptor is no longer engaged by its D(d) ligand. In contrast, Ly49A+ NK cells from D(d) x MxCre double Tg mice were unable to react to D(d)-negative cells. These Ly49A+ NK cells retained reactivity to target cells that were completely devoid of MHC class I molecules, suggesting that they were not anergic. Variegated D(d) expression thus impacts specifically missing D(d) but not globally missing class I reactivity by Ly49A+ NK cells. We propose that the absence of D(d) from some host cells results in the acquisition of only partial missing self-reactivity.

DNA methylation profiles of human active and inactive X chromosomes.

X-chromosome inactivation (XCI) is a dosage compensation mechanism that silences the majority of genes on one X chromosome in each female cell. To characterize epigenetic changes that accompany this process, we measured DNA methylation levels in 45,X patients carrying a single active X chromosome (X(a)), and in normal females, who carry one X(a) and one inactive X (X(i)). Methylated DNA was immunoprecipitated and hybridized to high-density oligonucleotide arrays covering the X chromosome, generating epigenetic profiles of active and inactive X chromosomes. We observed that XCI is accompanied by changes in DNA methylation specifically at CpG islands (CGIs). While the majority of CGIs show increased methylation levels on the X(i), XCI actually results in significant reductions in methylation at 7% of CGIs. Both intra- and inter-genic CGIs undergo epigenetic modification, with the biggest increase in methylation occurring at the promoters of genes silenced by XCI. In contrast, genes escaping XCI generally have low levels of promoter methylation, while genes that show inter-individual variation in silencing show intermediate increases in methylation. Thus, promoter methylation and susceptibility to XCI are correlated. We also observed a global correlation between CGI methylation and the evolutionary age of X-chromosome strata, and that genes escaping XCI show increased methylation within gene bodies. We used our epigenetic map to predict 26 novel genes escaping XCI, and searched for parent-of-origin-specific methylation differences, but found no evidence to support imprinting on the human X chromosome. Our study provides a detailed analysis of the epigenetic profile of active and inactive X chromosomes.

Inactivation of PPARβ/δ adversely affects satellite cells and reduces postnatal myogenesis.

Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a ubiquitously expressed gene with higher levels observed in skeletal muscle. Recently, our laboratory showed (Bonala S, Lokireddy S, Arigela H, Teng S, Wahli W, Sharma M, McFarlane C, Kambadur R. J Biol Chem 287: 12935-12951, 2012) that PPARβ/δ modulates myostatin activity to induce myogenesis in skeletal muscle. In the present study, we show that PPARβ/δ-null mice display reduced body weight, skeletal muscle weight, and myofiber atrophy during postnatal development. In addition, a significant reduction in satellite cell number was observed in PPARβ/δ-null mice, suggesting a role for PPARβ/δ in muscle regeneration. To investigate this, tibialis anterior muscles were injured with notexin, and muscle regeneration was monitored on days 3, 5, 7, and 28 postinjury. Immunohistochemical analysis revealed an increased inflammatory response and reduced myoblast proliferation in regenerating muscle from PPARβ/δ-null mice. Histological analysis confirmed that the regenerated muscle fibers of PPARβ/δ-null mice maintained an atrophy phenotype with reduced numbers of centrally placed nuclei. Even though satellite cell numbers were reduced before injury, satellite cell self-renewal was found to be unaffected in PPARβ/δ-null mice after regeneration. Previously, our laboratory had showed (Bonala S, Lokireddy S, Arigela H, Teng S, Wahli W, Sharma M, McFarlane C, Kambadur R. J Biol Chem 287: 12935-12951, 2012) that inactivation of PPARβ/δ increases myostatin signaling and inhibits myogenesis. Our results here indeed confirm that inactivation of myostatin signaling rescues the atrophy phenotype and improves muscle fiber cross-sectional area in both uninjured and regenerated tibialis anterior muscle from PPARβ/δ-null mice. Taken together, these data suggest that absence of PPARβ/δ leads to loss of satellite cells, impaired skeletal muscle regeneration, and postnatal myogenesis. Furthermore, our results also demonstrate that functional antagonism of myostatin has utility in rescuing these effects.

Evolution of dosage compensation under sexual selection differs between X and Z chromosomes.

Complete sex chromosome dosage compensation has more often been observed in XY than ZW species. In this study, using a population genetic model and the chicken transcriptome, we assess whether sexual conflict can account for this difference. Sexual conflict over expression is inevitable when mutation effects are correlated across the sexes, as compensatory mutations in the heterogametic sex lead to hyperexpression in the homogametic sex. Coupled with stronger selection and greater reproductive variance in males, this results in slower and less complete evolution of Z compared with X dosage compensation. Using expression variance as a measure of selection strength, we find that, as predicted by the model, dosage compensation in the chicken is most pronounced in genes that are under strong selection biased towards females. Our study explains the pattern of weak dosage compensation in ZW systems, and suggests that sexual selection plays a major role in shaping sex chromosome dosage compensation.

Short stature and primary ovarian insufficiency possibly due to chromosomal position effect in a balanced X;1 translocation.

BACKGROUND: Primary ovarian insufficiency (POI) is defined as a primary ovarian defect characterized by absent menarche (primary amenorrhea), a decrease in the initial primordial follicle number, high follicle-stimulating hormone (FSH) levels and hypoestrogenism. Although the etiology of a majority of POI cases is not yet identified, several data suggest that POI has a strong genetic component. Conventional cytogenetic and molecular analyses have identified regions of the X chromosome that are associated with ovarian function, as well as POI candidate genes, such as FMR1 and DIAPH2. Here we describe a 10.5-year-old girl presenting with high FSH and luteinizing hormone (LH) levels, pathologic GH stimulation arginine and clonidine tests, short stature, pterygium, ovarian dysgenesis, hirsutism and POI. RESULTS: Cytogenetic analysis demonstrated a balanced reciprocal translocation between the q arms of chromosomes X and 1, with breakpoints falling in Xq21 and 1q41 bands. Molecular studies did not unravel any chromosome microdeletion/microduplication, and no XIST-mediated inactivation was found on the derivative chromosome 1. Interestingly, through immunofluorescence assays, we found that part of the Xq21q22 trait, translocated to chromosome 1q41, was late replicating and therefore possibly inactivated in 30 % metaphases both in lymphocytes and skin fibroblasts, in addition to a skewed 100 % inactivation of the normal X chromosome. These findings suggest that a dysregulation of gene expression might occur in this region. Two genes mapping to the Xq translocated region, namely DIAPH2 and FMR1, were found overexpressed if compared with controls. CONCLUSIONS: We report a case in which gonadal dysgenesis and POI are associated with over-expression of DIAPH2 gene and of FMR1 gene in wild type form. We hypothesize that this over-expression is possibly due to a phenomenon known as "chromosomal position effect", which accounts for gene expression variations depending on their localization within the nucleus. For the same effect a double mosaic inactivation of genes mapping to the Xq21-q22 region, demonstrated by immunofluorescence assays, may be the cause of a functional Xq partial monosomy leading to most Turner traits of the proband's phenotype.

Simultaneous allergen inactivation and detoxification of castor bean cake by treatment with calcium compounds

Ricinus communis L. is of great economic importance due to the oil extracted from its seeds. Castor oil has been used for pharmaceutical and industrial applications, as a lubricant or coating agent, as a component of plastic products, as a fungicide or in the synthesis of biodiesel fuels. After oil extraction, a castor cake with a large amount of protein is obtained. However, this by-product cannot be used as animal feed due to the presence of toxic (ricin) and allergenic (2S albumin) proteins. Here, we propose two processes for detoxification and allergen inactivation of the castor cake. In addition, we establish a biological test to detect ricin and validate these detoxification processes. In this test, Vero cells were treated with ricin, and cell death was assessed by cell counting and measurement of lactate dehydrogenase activity. The limit of detection of the Vero cell assay was 10 ng/mL using a concentration of 1.6 x 10(5) cells/well. Solid-state fermentation (SSF) and treatment with calcium compounds were used as cake detoxification processes. For SSF, Aspergillus niger was grown using a castor cake as a substrate, and this cake was analyzed after 24, 48, 72, and 96 h of SSF. Ricin was eliminated after 24 h of SSF treatment. The cake was treated with 4 or 8% Ca(OH)2 or CaO, and both the toxicity and the allergenic properties were entirely abolished. A by-product free of toxicity and allergens was obtained.

Deiodinase-mediated thyroid hormone inactivation minimizes thyroid hormone signaling in the early development of fetal skeleton

Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To Study this, pregnant once were made hypothyroid and fetuses harvested at embryonic days (E) 12.5, 14.5, 16.5 and 18.5. Despite a marked reduction in fetal tissue concentration of both T4 and T3, bone development, as assessed at the distal epiphyseal growth plate of the femur and vertebra, was largely preserved Lip to E16.5. Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrophic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocyres, in addition to a slight delay in endochondral and intramembranous ossification. This Suggests that LIP to E 16.5, thyroid hormone signaling in bone is kept to a minimum. In fact, measuring the expression level of the activating and inactivating iodothyronine deiodinases (D2 and D3) helped understand how this is achieved. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly (similar to 10-fold) at E18.5, and even more at 14 days after birth (P14). In contrast. D2 mRNA expression increased significantly by E18.5 and markedly (similar to 2.5-fold) by P14. The reciprocal expression levels of D2 and D3 genes during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time Suggest that coordinated reciprocal deiodinase expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development. (c) 2008 Elsevier Inc. All rights reserved.

Isolation and characterization of latent and active polyphenoloxidase in BRS Clara (CNPUV 154-147 x Centennial seedless) and BRS Morena (Marroo seedless x Centennial seedless) seedless table grapes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Inactivation and disposal of by-products from Mycobacterium smegmatis by photoelectrocatalytic oxidation using Ti/TiO2-Ag nanotube electrodes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A new class of neurotoxin from wasp venom slows inactivation of sodium current

The effects of alpha-pompilidotoxin (alpha-PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that alpha-PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action or alpha-PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and alpha-scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of alpha-PMTX on Na+ channels by whole-cell recordings from rat trigeminal neurons. We found that alpha-PMTX stowed the Na+ channels inactivation process without changing the peak current-voltage relationship or the activation time course of tetrodotoxin (TTX)-sensitive Na+ currents, and that alpha-PMTX had voltage-dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that alpha-PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of alpha-PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein.

DUPLICATION OF THE SHORT ARM OF THE X-CHROMOSOME IN MOTHER AND DAUGHTER

An 11-year-old girl with short stature, mental retardation, and mild dysmorphic features was found to have an inverted duplication of most of the short arm of the X chromosome [dic inv dup(X)(qter --> p22.3 = p22.3 --> cen:)]. Her mother, who is also short and retarded, carries the same duplication. Fluorescence in situ hybridization with an X chromosome library, and with X centromere-specific alpha satellite and telomere probes, was useful in characterizing the duplication. In most females with structurally abnormal X chromosomes, the abnormal chromosome is inactivated. Although the duplicated X was consistently late replicating in the mother, X chromosome inactivation studies in the proband indicated that in 11 % of her lymphocytes the duplicated X was active.